Human thyroxine-binding globulin (TBG) has been isolated in milligram quantities in highly purified form with retention of high binding affinity. The maximal binding capacity of the TBG for thyroxine (T4) and triiodothyronine (T3) was about 0.5 mole hormone per mole protein as determined by fluorescence quenching and ultrafiltration. As measured by ultrafiltration, the binding constant of TBG for T4 was 2.8-3.8 x 10 to the 9th power moles/liter, at 23-24 degrees C in 0.06 M potassium phosphate bufferr, pH 7.4. Under similar conditions, the binding constant for T3 was 3.3-6.3 x 10 to the 8th power moles/liter. T4 seems to be bound about 6-8 times more tightly than T3 by TBG. Chemical analysis of TBG by gas liquid chromatography revealed the presence of long chain fatty acids on the protein. Palmic acid was present in greatest amount followed by oleic, hydroxypalmitic and stearic acid. Animal experiments performed with perfused rat livers after in vivo injection of trace quantities of I125-T3 indicated the presence of at least three T3-binding proteins in liver cytosol (soluble supernatant after centrifugation of homogenate at 100,000 x g). As ascertained by chromatography on Sephadex the molecular weights of these proteins were in the regions of 400,000, 80-100,000 and a smaller molecular weight protein of less than 50,000.